The genus Brassica includes one of the world""s most important oilseed crops, canola. Considerable effort has been made to improve its agronomic qualities by selective breeding techniques. The goals for improving this crop include increased yield and disease resistance, in addition to altering the oil content and composition.
The present invention provides a novel Brassica napus oleifera annua variety, an agronomically superior high oleic canola variety having a unique fatty acid profile, designated xe2x80x9cNex 705xe2x80x9d. Also provided in the present invention are seeds of canola variety Nex 705, plants produced from seeds of Nex 705, and tissue cultures of regenerable cells of the canola plant grown from Nex 705 seed.
As used herein, the following definitions are provided:
Definitions of Classification Terms for Variety Description of Canola Cultivar Nex 705
1.0 Classification:
1.1 Botanical and common name: The genus, species, and type define the botanical classification of the variety.
1.2 Type of variety: The type of variety defines the method of propagation of the variety as: Open pollinated (planted in isolation and plants allowed to inter mate), Hybrid (made by crossing two or more inbred parents), Self-pollinated (plants of the variety do not inter mate, but rather are completely self-pollinating), First generation synthetic (the variety sold is the product of the first generation of all possible intercrosses between a selected set of 3 or more inbred parents), Advanced generation synthetic (the variety sold is the product of repeated inter mating generations from a first generation synthetic).
2.0 Seasonal Type: Two seasonal variety types exist: Spring (planted in the spring to early summer, flower mid-summer and harvested in the late summer to fall) and Winter (planted in the late summer to fall, stay in a rosette form without flowering over the winter, flower late spring and harvested mid to late summer).
3.0 Characteristics of Plants before Flowering:
3.1 Cotyledon width: The cotyledons are leaf structures that form in the developing seeds of canola and which make up the majority of the mature seed of these species. When the seed germinates, the cotyledons are pushed out of the soil by the growing hypocotyl (segment of the seedling stem below the cotyledons and above the root) and they unfold as the first photosynthetic leafs of the plant. The width of the cotyledons varies by variety and can be classified as narrow, medium, or wide.
3.2 Seedling growth habit: The rosette consists of the first 2-8 true leaves and a variety can be characterized as having a strong rosette (closely packed leaves) or a weak rosette (loosely arranged leaves).
3.3 Stemxe2x80x94intensity of anthocyanin coloration: The stems and other organs of canola plants can have varying degrees of purple coloration which is due to the presence of anthocyanin (purple) pigments. The degree of coloration is somewhat subject to growing conditions, but varieties typically show varying degrees of coloration ranging from: absent (no purple)/very weak to very strong (deep purple coloration).
3.4 Leafxe2x80x94development of lobes: The leaves on the upper portion of the stem can show varying degrees of development of lobes which are disconnected from one another along the petiole of the leaf. The degree of lobing is variety specific and can range from absent (no lobes)/weak through very strong (abundant lobes).
3.5 Leafxe2x80x94indentation of margin: The leaves on the upper portion of the stem can also show varying degrees of serration along the leaf margins. The degree of serration or indentation of the leaf margins can vary from absent (smooth margin)/weak to strong (heavy saw-tooth like margin).
3.6 Leafxe2x80x94blade color: The color of the leaf blades is variety specific and can range from light to medium dark green to blue green.
3.7 Leafxe2x80x94pubescence: The leaf pubescence is the degree of hairiness of the leaf surface and is especially useful for distinguishing between the canola species. There are two main classes of pubescence which are glabrous (smooth/not hairy) and pubescent (hairy) which mainly differentiate between the B. napus and B. rapa species, respectively.
3.8 Leafxe2x80x94glaucosity: This refers to the waxiness of the leaves and is characteristic of specific varieties although environment can have some effect on the degree of waxiness. This trait can range from absent (no waxiness)/weak through very strong. The degree of waxiness can be best determined by rubbing the leaf surface and noting the degree of wax present.
3.9 Leafxe2x80x94attachment to the stem: This trait is especially useful for distinguishing between the two canola species. The base of the leaf blade of the upper stem leaves of B. rapa completely clasp the stem whereas those of the B. napus only partially clasp the stem. Those of the mustard species do not clasp the stem at all.
3.10 Leafxe2x80x94surface: The leaf surface can also be used to distinguish between varieties. The surface can be smooth or rugose (lumpy) with varying degrees between the two extremes.
4.0 Characteristics of Plants after Flowering:
4.1 Flowering date: This is measured by the number of days from planting to the stage when 50% of the plants in a population have one or more open flowers. This varies from variety to variety.
4.2 Plant height: This is the height of the plant at the end of flowering if the floral branches are extended upright (i.e., not lodged). This varies from variety to variety and although it can be influenced by environment, relative comparisons between varieties grown side by side are useful for variety identification.
4.3 Growth habit: At the end of flowering, the angle relative to the ground surface of the outermost fully expanded leaf petioles is a variety specific trait. This trait can range from erect (very upright along the stem) to prostrate (almost horizontal and parallel with the ground surface).
4.4 Flower budsxe2x80x94location: The location of the unopened flower buds relative to the adjacent opened flowers is useful in distinguishing between the canola species. The unopened buds are held above the most recently opened flowers in B. napus and they are positioned below the most recently opened flower buds in B. rapa. 
4.5 Petal color: The petal color on the first day a flower opens can be a distinguishing characteristic for a variety. It can be white, varying shades of yellow or orange.
4.6 Anther dotting: The presence/absence of anther dotting (colored spots on the tips of anthers) and if present, the percentage of anther dotting on the tips of anthers in newly opened flowers is also a distinguishing trait for varieties.
4.7 Anther arrangement; The orientation of the anthers in fully opened flowers can also be useful as an identifying trait. This can range from introse (facing inward toward pistil), erect (neither inward not outward), or extrose (facing outward away from pistil).
4.8 Anther fertility: This is a measure of the amount of pollen produced on the anthers of a flower. It can range from sterile (such as in female parents used for hybrid seed production) to fertile (all anthers shedding).
4.9 Silique (pod)xe2x80x94type: This is typically a bilateral single pod for both species of canola and is not really useful for variety identification within these species.
4.10 Silique (pod)xe2x80x94length: This is the length of the fully developed pods and can range from short to medium to long. It is best used by making comparisons relative to reference varieties.
4.11 Silique (pod)xe2x80x94width: This is the width of the fully developed pods and can range from narrow to medium to wide. It is best used by making comparisons relative to reference varieties.
4.12 Silique (pod)xe2x80x94habit: This is also a trait which is variety specific and is a measure of the orientation of the pods along the racemes (flowering stems). This trait can range from erect (pods angled close to racemes) through horizontal (pods perpendicular to racemes) through arching (pods show distinct arching habit).
4.13 Silique (pod)xe2x80x94length of beak: The beak is the segment at the end of the pod which does not contain seed (it is a remnant of the stigma and style for the flower). The length of the beak can be variety specific and can range form short through medium through long.
4.14 Silique (pod)xe2x80x94length of pedicel: The pedicel is the stem that attaches the pod to the raceme of flowering shoot. The length of the-pedicel can be variety specific and can vary from short through medium through long.
4.15 Maturity: The maturity of a variety is measured as the number of days between planting and physiological maturity. This is useful trait in distinguishing varieties relative to one another.
5.0 Seed Characteristics:
5.1 Seed coatxe2x80x94color: The color of the seed coat can be variety specific and can range from black through brown through yellow. Color can also be mixed for some varieties. 5.2 Seed coatxe2x80x94mucilage: This is useful for differentiating between the two species of canola with B. rapa varieties having mucilage present in their seed coats whereas B. napus varieties do not have this present. It is detected by imbibing seeds with water and monitoring the mucilage that is exuded by the seed.
6.0 Agronomic Characteristics:
6.1 Resistance to lodging: This measures the ability of a variety to stand up in the field under high yield conditions and severe environmental factors. A variety can have good (remain upright), fair, or poor (falls over) resistance to lodging. The degree of resistance to lodging is not expressed under all conditions but is most meaningful when there is some degree of lodging in a field trial.
7.0 Reaction to Diseases: The reaction of varieties to various diseases which affect canola can also be useful in differentiating varieties. The two main diseases rated in canola are blackleg (causes lesions in the lower stems which cut off water and nutrient flow in the stem) and white rust (only a problem in the B. rapa species).
8.0 Quality Characteristics:
8.1 Oil content: This is measured as percent of the whole dried seed and is characteristic of different varieties. It can be determined using various analytical techniques such as NMR, NIR, and Soxhiet extraction.
8.2 Percentage of total fatty acids: This is determined by extracting a sample of oil from seed, producing the methyl esters of fatty acids present in that oil sample and analyzing the proportions of the various fatty acid in the sample using gas chromatography. The fatty acid composition can also be a distinguishing characteristic of a variety.
8.3 Maximum amount of erucic acid allowed in foundation seed of this variety for pedigreed seed purity determination. The level of erucic acid in foundation seed lots is determined to arrive at this figure which is useful for purity determinations.
8.4 Protein content: This is measured as percent of whole dried seed and is characteristic of different varieties. This can be determined using various analytical techniques such as NIR and Kjeldahl.
8.5 Glucosinolates: These are measured in micromoles (xcexcm) of total alipathic glucosinolates per gram of air-dried oil-free meal. The level of glucosinolates is somewhat influenced by the sulfur fertility of the soil, but is also controlled by the genetic makeup of each variety and thus can be useful in characterizing varieties.
Spring canola variety Nex 705 (xe2x80x9cNex 705xe2x80x9d) is an agronomically superior, high oleic canola variety (Brassica napus oleifera annua) developed at the Agrigenetics Madison laboratories by microspore culture from the cross Cyclone (canola) X AG021 (high oleic canola).
Development of the Invention: H080-01, a high (75%) oleic mutant of the canola variety Regent, was developed by SunGene Technologies using chemical mutagenesis (U.S. patent application Ser. No. 518,669, now abandoned). The high oleic acid trait from H080-01 was combined with the low linolenic acid trait from the germplasm accession BN0010 by crossing these two lines at the Agrigenetics Madison laboratories. A F3 single plant selection from this cross, 90W114-0412-88, was identified with 80.5% oleic acid and 1.3% linolenic acid. For reference, the Agrigenetics high oleic acid/low linolenic acid Canola variety AG019 arose from a different F3 single plant selection from this same cross (AG019 is the subject of issued U.S. Pat. No. 5,965,755). 90W114-04-12-88 was crossed as male parent onto the spring Canola variety Global (cross designation 91S070. A F3 half-seed selection from this cross, 91S0070-18-117-143, was identified with 80.1% oleic acid and 1.8% linolenic acid. The plant arising from this F3 half-seed was crossed as male to the spring canola variety Global (BC1 to Global, cross designation 93S090. A F2 half-seed selection from this cross, 93S090-5-419, was identified with 80.7% oleic acid and 1.9% linolenic acid. This single F2 plant selection was increased as the high oleic acid/low linolenic acid strain SVO95-08 arose from different F2 half-seed selection from this same cross and is being used for commercial production of high oleic acid/low linolenic acid Canola oil under the variety name xe2x80x9cNex-700xe2x80x9d. SVO 95-09 was crossed as male onto the high oil, partially yellow seeded spring Canola variety Polo (cross designation 94S061. Microspore culture with in-vitro doubling of chromosome number was conducted on F1 plants on the cross 94S061 to produce 174 dihaploid progenies. Bulk seed samples from these dihaploid progenies were screened for fatty acid composition in order to select those progenies with high oleic acid and/or low linolenic acid. These dihaploid progenies were also rated for seed color, as the high oil content from Polo is associated with a yellow or yellow-brown seed color. In total, 65 dihaploid progenies with high oleic acid and/or low linolenic acid were identified and seed of each of these was increased at the Agrigenetics Woodland station. These 65 dihaploid progenies were evaluated in preliminary yield trials at 2 locations in western Canada. Based on the results of these trials, a dihaploid strain 94S061-DH13 (designated xe2x80x9cM94S007xe2x80x9d) was identified as having high yield, high oil content and high oleic acid/low linolenic acid levels. This strain was further multiplied and entered into Canadian contract registration trials in which it continued to perform extremely well. M94S007 entered into megaplot trials and performed extremely well also.
The subject novel canola variety can be genetically engineered to include a selected gene (gene of interest) using known transformation methods routine in the art such that expression of the gene of interest is observed. Methods of transformation include use of Agrobacterium, viral vectors, microinjection, PEG, biolistics, and electroporation which are all routinely used to introduce foreign DNA into plant cells. Once in the cell, the foreign DNA incorporates into the plant genome. In a preferred embodiment, the transformation contemplates constructing a vector comprising plant regulatory sequences and a gene(s) of interest, placing the vector into a selected strain of Agrobacterium, and infecting selected plant cells with the Agrobacterium under conditions sufficient to result in transfer of at least some of the vectors from the Agrobacterium to the plant cells, whereby the gene of interest is expressed in the plant cells. Regulatory sequences can include both promoter and termination sequences.
Possible regulatory sequences include, but are not limited to, any promoter already shown to be constitutive for expression, such as those of viral origin (CaMV 19S and 35S, TMV, AMV) or so-called xe2x80x9chousekeepingxe2x80x9d genes (ubiquitin, actin, tubulin) with their corresponding termination/poly A+ sequences. Also, seed-and/or developmentally-specific promoters, such as those from plant fatty acid/lipid biosynthesis genes (ACPs, acyltransferases, desaturases, lipid transfer protein genes) or from storage protein genes (zein, napin, cruciferin, conglycinin, or lectin genes, for example), with their corresponding termination/poly A+ sequences can be used for targeted expression. In addition, the gene can be placed under the regulation of inducible promoters and their termination sequences so that gene expression is induced by light (rbcS-3A, cab-1), heat (hsp gene promoters) or wounding (mannopine, HGPGs). It is clear to one skilled in the art that a promoter may be used either in native or truncated form, and may be paired with its own or a heterologous termination/polyA+ sequence, and enhancers as are known in the art.
In addition, expressed gene products may be localized to specific organelles in the plant cell by ligating DNA coded for peptide leader sequences to the gene of interest. Such leader sequences can be obtained from several genes of either plant or other sources. These genes encode cytoplasmically-synthesized proteins directed to, for example, mitochondria (the F1-ATPase beta subunit from yeast or tobacco, cytochrome c1 from yeast), chloroplasts (cytochrome oxidase subunit Va from yeast, small subunit of rubisco from pea), endoplasmic reticulum lumen (protein disulfide isomerase), vacuole (carboxypeptidase Y and proteinase A from yeast, phytohemagglutinin from French bean), peroxisomes (D-aminoacid oxidase, uricase) and lysosomes (hydrolases).
A selectable marker gene for optimum transformation selection is preferably employed. Such markers are typically genes which encode resistance to various toxic chemicals such as antibiotics and herbicides; the resistance is usually conferred by enzymes which typically render the chemical non-toxic to the transgenic plant. Such toxic chemicals include, for example, hygromycin, kanamycin, methotrexate, phosphinothricin, glyphosate, and bromoxynil. Enzymes which confer resistance to these chemicals are hygromycin phosphotransferase, neomycin phosphotransferase, dihydrofolate reductase, phosphinthricin acetyl transferase, ESPS, and AHAS. Genes which code for resistance are well known to those of ordinary skill in the art of plant transformation. Plants transformed with such genes are able to grow in the presence of the toxic compound, while non-transformed plants are not. Therefore, such genes serve both as a means of selecting transformed plants and as a marker for transformation, indicating that transformation has occurred.
Plant tissue useful in accord with the teachings herein includes, but is not limited to, leaves, hypocotyls, cotyledons, stems, callus, single cells that contain a biological control agent gene, and protoplasts.
In a particular embodiment, transformed callus tissue is selected by growth on selection medium (e.g., medium which contains a toxic chemical and for which the transformed plant contains a resistance gene, by virtue of its transformation). Transformed plants are regenerated and screened for the presence of the expressed gene product. This involves analyzing tissue by at least one molecular or biological assays to determine which, if any, transformants contained the biocontrol agent polynucleotides or proteins. These assays include assays of the tissue for the expression of the resistance gene enzyme, and assays of the tissue for the presence of control agent polynucleotide by, for example, a Southern assay or a PCR assay.
Those plants which are positive for the gene of interest are grown to maturity, and tissue can be analyzed for expression of the gene of interest by looking for the polypeptide encoded by the polynucleotide, as for example via a Western blot analysis.
It is now well known in the art that when synthesizing a gene for improved expression in a host cell it is desirable to design the gene such that its frequency of codon usage approaches the frequency of codon usage of the host cell. For purposes of the subject invention, xe2x80x9cfrequency of preferred codon usagexe2x80x9d refers to the preference exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. To determine the frequency of usage of a particular codon in a gene, the number of occurrences of that codon in the gene is divided by the total number of occurrences of all codons specifying the same amino acid in the gene. Similarly, the frequency of preferred codon usage exhibited by a plant cell can be calculated by averaging frequency of preferred codon usage in a large number of genes expressed by the plant cell. It is preferable that this analysis be limited to genes that are highly expressed by the host cell. Alternatively, the gene may be synthesized to have a greater number of the host""s most preferred codon for each amino acid, or to reduce the number of codons that are rarely used by the host.
Thus, in one embodiment of the subject invention, plant cells can be genetically engineered, e.g., transformed with genes to attain desired expression levels. To provide genes having enhanced expression, the DNA sequence of the gene can be modified to comprise codons preferred by highly expressed genes to attain an A+T content in nucleotide base composition which is substantially that found in the transformed host cell. It is also preferable to form an initiation sequence optimal for said plant cell, and to eliminate sequences that cause destabilization, inappropriate polyadenylation, degradation and termination of RNA, and to avoid sequences that constitute secondary structure hairpins and RNA splice sites. For example, in synthetic genes, the codons used to specify a given amino acid can be selected with regard to the distribution frequency of codon usage employed in highly expressed genes in the plant cell to specify that amino acid. As is appreciated by those skilled in the art, the distribution frequency of codon usage utilized in the synthetic gene is a determinant of the level of expression.